The present invention relates to a diagnostic reagent and a method for diagnosing Crohn""s disease.
Autoimmune diseases refer to a phenomenon wherein a biological defense system (immune system) attacks the cells of its own. Antibodies and lymphocytes reactive with the autoantigen are derived, which in turn develops tissue disorders and lesions. The autoimmune diseases are generally divided into two groups: those without organ specificity and those with organ specificity. The mechanism of the onset of the autoimmune diseases is mostly unclear, though involvement of autoimmunity is suggested. There are many problems to be solved such as diagnosis method and the like, which include the mechanism of the onset of autoimmune diseases.
The autoimmune diseases include Crohn""s disease, as one of the diseases whose etiology has not been elucidated, in which an immune reaction against autoantigen and allergy are considered to be involved. This disease is an inflammatory bowel disease associated with inflammatory changes throughout the full thickness of the wall of the digestive tract, discontinuous deep ulcer and histologically noncaseating granuloma. The skipping of the lesion also characterizes this disease. The definite diagnosis of Crohn""s disease is based on a comprehensive observation of the disease state, X rays, endoscopy and tissue images. However, since the diagnosis is possible only after the progress of the disease, earlier diagnosis is desired. In addition, differential diagnosis from other inflammatory bowel diseases, such as acute or chronic appendicitis, tuberculosis of the intestine, ulcerative colitis, ischemic enteritis and the like, is required.
There are reports documenting that the genes of membrane proteins such as interleukin 2 (IL-2) receptor, transferrin receptor, E-selectin (also referred to as ELAM-1, endothelial leukocyte adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule-1), L-selectin, CD11, OX40, OX40 ligand and the like, and cytokines and chemokines such as interleukin 1xcex2 (IL-1xcex2), IL-2, interleukin 6 (IL-6), interleukin 15 (IL-15), tumor necrosis factor a (TNF-xcex1), interleukin 18 (IL-18), interleukin 8 (IL-8), MCP-1, ENA-78 and the like are up-regulated in Crohn""s disease, from a molecular biological approach taking note of the expression of cytokines and adhesion molecules. However, most of these genes in the reports showed the up-regulation due to a non-specific immune response associated with the inflammation observed in Crohn""s disease, and the up-regulation is not specific to Crohn""s disease.
It is therefore an object of the present invention to provide a method useful for diagnosing Crohn""s disease and a reagent for the diagnosis.
In view of the above-mentioned problems, the present inventors have conducted intensive studies of the expression profiles at the gene level of Crohn""s disease, in an attempt to enable early diagnosis of Crohn""s disease and differential diagnosis from other diseases. Since a lesion and a non-lesion part can be clearly distinguished visually in Crohn""s disease, a differential display method (Liang, P., and Pardee, A. B. Science 257:967-971 (1992), Liang, P., and Pardee, A. B. Curr. Opin. Immunol. 7:274-280 (1995)), wherein the genes expressed in the lesion and the non-lesion part in the same individual can be compared, was employed to compare gene expression profiles in the lesion and the non-lesion part. As a result, the expression of a certain kind of gene in the lesion was found to have been specifically potentiated and the gene could be identified, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) A reagent for diagnosing Crohn""s disease, which contains at least one member selected from the group consisting of (i) a substance having a specific affinity for a gene of a type 6 protein phosphatase regulated by interleukin 2 (type 6 protein phosphatase regulated by IL-2; hereinafter to be also referred to as PP6 regulated by IL-2), (ii) a substance having a specific affinity for a gene of a Traf 2 and Nck interacting kinase (hereinafter to be also referred to as TNIK), (iii) a substance having a specific affinity for a gene of a FLICE inhibitory protein (hereinafter to be also referred to as FLIP), and (iv) a substance having a specific affinity for a gene of a glucocorticoid receptor xcex1 (hereinafter to be also referred to as GRxcex1).
(2) The reagent for diagnosing Crohn""s disease according to the above-mentioned mentioned (1), which further contains at least one member selected from the group consisting of (v) a substance having a specific affinity for a cytochrome oxidase subunit I gene and (vi) a substance having a specific affinity for a cytochrome b gene.
(3) The reagent for diagnosing Crohn""s disease according to the above-mentioned (1) or (2), wherein the substance having a specific affinity is an oligonucleotide or polynucleotide probe, or an oligonucleotide or polynucleotide primer pair.
(4) A reagent for diagnosing Crohn""s disease, which contains at least one member selected from the group consisting of (i) a substance having a specific affinity for PP6 regulated by IL-2, (ii) a substance having a specific affinity for TNIK, (iii) a substance having a specific affinity for FLIP, and (iv) a substance having a specific affinity for GRxcex1.
(5) The reagent for diagnosing Crohn""s disease according to the above-mentioned (4), which further contains at least one member selected from the group consisting of (v) a substance having a specific affinity for a cytochrome oxidase subunit I and (vi) a substance having a specific affinity for cytochrome b.
(6) The reagent for diagnosing Crohn""s disease according to the above-mentioned (4) or (5), wherein the substance having a specific affinity is an antibody or a fragment thereof.
(7) A method for diagnosing Crohn""s disease, which comprises the steps of
(a) taking a biological sample from an animal that developed or is associated with a risk of developing Crohn""s disease, and
(b) analyzing the expression of at least one gene selected from the group consisting of a gene of PP6 regulated by IL-2, a TNIK gene, a FLIP gene and a GRxcex1 gene, in a biological sample thereof.
(8) The method for diagnosing Crohn""s disease according to the above-mentioned (7), which further includes analyzing the expression of at least one gene selected from the group consisting of a cytochrome oxidase subunit I gene and a cytochrome b gene.
(9) A method for diagnosing Crohn""s disease, which comprises the steps of
(a) taking a biological sample from an animal that developed or is associated with a risk of developing Crohn""s disease, and
(b) analyzing the expression of at least one protein selected from the group consisting of PP6 regulated by IL-2, TNIK, FLIP and GRxcex1, in a biological sample thereof.
(10) The method for diagnosing Crohn""s disease according to the above-mentioned (9), which further includes analyzing the expression of at least one protein selected from the group consisting of cytochrome oxidase subunit I and cytochrome b.
(11) The method for diagnosing Crohn""s disease according to any of the above-mentioned (7) to (10), wherein the biological sample is an ileum tissue or colon tissue derived from the animal.
The gene in the present invention may be of any form unless otherwise particularly specified. For example, complementary DNA (cDNA) prepared from mRNA and the like are included besides mRNA.
The respective elements that may be contained in the diagnostic reagent according to the present invention are explained in detail in the following.
(i) PP6 Regulated by IL-2 [Type 6 Protein Phosphatase Regulated by IL-2; Protein (36 kDa) described in Filali, M., et al., J. Cell. Biochem. 73:153-163 (1999)]
The PP6 regulated by IL-2 is a phosphoprotein having a 98% homology to human PP6 at the amino acid level, and its expression is derived by IL-2 in the peripheral T cell. Its precise function has not been elucidated but involvement in the cell proliferation is suggested (Filali, M., et al., (1999) ibid.).
Examples of the substance having a specific affinity for the gene of PP6 regulated by IL-2, which is contained in the reagent for diagnosing Crohn""s disease of the present invention, include an oligonucleotide or polynucleotide probe (hereinafter to be conveniently referred to simply as a probe) having a specific affinity for the gene, and an oligonucleotide or polynucleotide primer pair (hereinafter to be conveniently referred to simply as a primer pair), wherein the specific affinity for the gene means the ability to specifically hybridize only to the objective gene. Therefore, the probe and the primer pair may be completely complementary to the entire gene or a part thereof, or may include one to several mismatches as long as they have the above-mentioned property. The probe and the primer pair are not subject to any particular limitation as long as they have specific affinity for the gene. Examples thereof include the entire nucleotide sequence of the gene or a part thereof, an oligonucleotide or polynucleotide containing a sequence complementary thereto, and the like, which may be selected as appropriate depending on the form of the gene to be detected. When PCR and the like are conducted as mentioned later using the diagnostic reagent of the present invention, the oligonucleotides depicted in SEQ ID NO. 1 and SEQ ID NO. 2 can be used as primer pairs. The origins of oligonucleotide and polynucleotide are not subject to any particular limitation as long as they have specific affinity for the gene. They may be synthesized or obtained by cleaving out the necessary portion from the gene, and purifying it according to a typical method. These oligonucleotide and polynucleotide may be labeled with a fluorescent substance, enzyme, radioisotope and the like.
Examples of the substance having a specific affinity for PP6 regulated by IL-2, which is contained in the reagent for diagnosing Crohn""s disease of the present invention, include an antibody having a specific affinity for the protein and a fragment thereof, wherein the specific affinity means the ability to specifically recognize the protein by an antigen-antibody reaction and to bind therewith. Such antibody and the fragment thereof are not subject to any particular limitation as long as they can specifically bind with the protein, and may be a polyclonal antibody, a monoclonal antibody or an operable fragment thereof. These antibodies and operable fragments thereof can be produced by a method generally employed in the pertinent field. When a polyclonal antibody is used, for example, an animal such as mouse and rabbit is immunized by injecting the protein subcutaneously to the back, intraperitoneally or into the vein and the like, and antiserum is harvested after increase in the antibody titer. When a monoclonal antibody is used, a hybridoma is prepared by a conventional method and a secretion thereby is taken. The antibody fragment is often produced by the expression, by a microorganism and the like, of a cloned gene fragment of an antibody. The purity of the antibody, antibody fragment and the like is not subject to any particular limitation as long as they can maintain the specific affinity for the protein. These antibodies and fragments thereof may be labeled with a fluorescent substance, enzyme, radioisotope and the like.
Furthermore, these may be obtained from the market.
(ii) TNIK [Traf2 and Nck Interacting Kinase; GCK Family Kinase described in Fu, C. A., et al., J. Biol. Chem. 274:30729-30737 (1999)]
The TNIK is a kinase that interacts with both Traf2 and Nck, and has been recently identified as a molecule that activates JNK.
Examples of the substance having a specific affinity for the TNIK gene, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include a probe and a primer pair having a specific affinity for the gene, wherein specific affinity for the gene means as mentioned above. The probe and the primer pair can be designed and modified based on the nucleotide sequence of the gene, as explained in the section for the above-mentioned PP6 regulated by IL-2. When PCR and the like are conducted using the diagnostic reagent of the present invention, the oligonucleotides depicted in SEQ ID NO. 3 and SEQ ID NO. 4 can be used as primer pairs.
Examples of the substance having a specific affinity for TNIK, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include an antibody having a specific affinity for the protein and a fragment thereof, wherein the specific affinity for the protein means as mentioned above. These antibodies and operable fragments thereof can be produced by a method similar to the general method explained in the section for the above-mentioned PP6 regulated by IL-2.
(iii) FLIP [FLICE inhibitory protein; described in Irmler, M., et al., Nature 388:190-195 (1997), Human FLIPL: GenBank Accession No. U97074, human FLIPS: GenBank Accession No. U97075]
The FLIP is a structural analog of FLICE, which is reported to suppress apoptosis by inhibiting association of FADD and FLICE (Irmler, M., et al., (1997) ibid., Hu, S., et al., J. Biol. Chem. 272:17255-17257 (1997)). It includes a long form (FLIPL) and a short form (FLIPs).
Examples of the substance having a specific affinity for the FLIP gene, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include a probe and a primer pair having a specific affinity for the gene, wherein the specific affinity for the gene means as mentioned above. The probe and the primer pair can be designed and modified based on the nucleotide sequence of the gene, as explained in the section for the above-mentioned PP6 regulated by IL-2. When PCR and the like are conducted using the diagnostic reagent of the present invention, the oligonucleotides depicted in SEQ ID NO. 5 and SEQ ID NO. 6 can be used as primer pairs for FLIPL, and those depicted in SEQ ID NO. 7 and SEQ ID NO. 8 for FLIPs.
Examples of the substance having a specific affinity for FLIP, which is contained in the reagent for diagnosing crohn""s disease according to the present invention, include an antibody having a specific affinity for the protein and a fragment thereof, wherein the specific affinity for the protein means as mentioned above. These antibodies and operable fragments thereof can be produced by a method similar to the general method explained in the section for the above-mentioned PP6 regulated by IL-2.
(iv) GRxcex1 [Glucocorticoid Receptor xcex1; Protein (94 kDa) described in Hollenberg, S. M., et al., Nature 318:635-641 (1985)]
The GRxcex1 is a receptor belonging to a nuclear receptor superfamily, wherein ligand is glucocorticoid, and is a transcriptional regulatory factor that promotes transcription of the target gene in a ligand-dependent manner.
Examples of the substance having a specific affinity for the GRxcex1 gene, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include a probe and a primer pair having a specific affinity for the gene, wherein the specific affinity for the gene means as mentioned above. The probe and the primer pair can be designed and modified based on the nucleotide sequence of the gene, as explained in the section for the above-mentioned PP6 regulated by IL-2. When PCR and the like are conducted using the diagnostic reagent of the present invention, the oligonucleotides depicted in SEQ ID NO. 9 and SEQ ID NO. 10 can be used as primer pairs.
Examples of the substance having a specific affinity for GRxcex1, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include an antibody having a specific affinity for the protein and a fragment thereof, wherein the specific affinity for the protein means as mentioned above. These antibodies and operable fragments thereof can be produced by a method similar to the general method explained in the section for the above-mentioned PP6 regulated by IL-2.
(v) Cytochrome Oxidase Subunit I [described in Sanger, F., et al., J. Mol. Biol. 143(2), 161-178 (1980), Anderson, S., et al., Nature 290 (5806), 457-465 (1981)]
The cytochrome oxidase is a terminal oxidase of an electron transfer system present in the mitochondrial inner membrane, and consists of 7 to 13 subunits. This enzyme is essential for synthesizing ATP from ADP and inorganic phosphorus. The NO produced in the inflamed area is known to bind with the cytochrome oxidase subunit I competitively with an oxygen molecule.
Examples of the substance having a specific affinity for the cytochrome oxidase subunit I gene, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include a probe and a primer pair having a specific affinity for the gene, wherein the specific affinity for the gene means as mentioned above. The probe and the primer pair can be designed and modified based on the nucleotide sequence of the gene, as explained in the section for the above-mentioned PP6 regulated by IL-2. When PCR and the like are conducted using the diagnostic reagent of the present invention, the oligonucleotides depicted in SEQ ID NO. 11 and SEQ ID NO. 12 can be used as primer pairs.
Examples of the substance having a specific affinity for cytochrome oxidase subunit I, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include an antibody having a specific affinity for the protein and a fragment thereof, wherein the specific affinity for the protein means as mentioned above. These antibodies and operable fragments thereof can be produced by a method similar to the general method explained in the section for the above-mentioned PP6 regulated by IL-2.
(vi) Cytochrome b [described in Anderson, S., et al., Nature 290 (5806), 457-465 (1981)]
The cytochrome refers to a group of heme protiens responsible for the electron transfer. The cytochrome b is present in a mitochondrial inner membrane along with c1, a3 and the like, and constitutes an electron transfer system.
Examples of the substance having a specific affinity for the cytochrome b gene, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include a probe and a primer pair having a specific affinity for the gene, wherein the specific affinity for the gene means as mentioned above. The probe and the primer pair can be designed and modified based on the nucleotide sequence of the gene, as explained in the section for the above-mentioned PP6 regulated by IL-2. When PCR and the like are conducted using the diagnostic reagent of the present invention, the oligonucleotides depicted in SEQ ID NO. 13 and SEQ ID NO. 14 can be used as primer pairs.
Examples of the substance having a specific affinity for cytochrome b, which is contained in the reagent for diagnosing Crohn""s disease according to the present invention, include an antibody having a specific affinity for the protein and a fragment thereof, wherein the specific affinity for the protein means as mentioned above. These antibodies and operable fragments thereof can be produced by a method similar to the general method explained in the section for the above-mentioned PP6 regulated by IL-2.
The respective elements (the above-mentioned (i)-(vi)), which are contained in the reagent for diagnosing Crohn""s disease according to the present invention, can be used alone. Preferably, the reagent contains at least one of or all of the above-mentioned (i) to (iv) having higher specificity to Crohn""s disease, i.e., a substance having a specific affinity for PP6 regulated by IL-2 and a gene of PP6 regulated by IL-2, a substance having a specific affinity for TNIK and its gene, a substance having a specific affinity for FLIP and its gene, and a substance having a specific affinity for GRxcex1 and its gene. Where desired, it may contain at least one of the above-mentioned (v) and (vi), i.e., a substance having a specific affinity for cytochrome oxidase subunit I and its gene and a substance having a specific affinity for cytochrome b and its gene. When plural substances are used, they may be admixed and used as one reagent or may be used as separate reagents. Even when plural substances are admixed and used as one reagent, it can easily distinguish each expression, based on the molecular weight of the objective protein or the length of the objective gene. However, particularly when the gene expression profiles of Crohn""s disease (diagnostic subjects) show interindividual differences, and when a quick and easy diagnosis of Crohn""s disease is desired, respective substances are preferably admixed and used as a single reagent. When a diagnosis including a detailed future treatment policy is desired, a diagnostic reagent containing one of the elements is preferably used.
The present invention also provides a method for diagnosing Crohn""s disease. The diagnostic method of the present invention is preferably applied using the aforementioned diagnostic reagent for Crohn""s disease. To be specific, a biological sample is first taken from an animal to be a diagnosis target. In this specification, by the xe2x80x9canimalxe2x80x9d is meant various mammals inclusive of human and birds. Examples thereof include human, monkey, dog, cat, cow, horse, pig, mouse, rabbit, chicken and the like. The biological sample is not subject to any particular limitation as long as it affords observation of noticeable changes in the expression of the above-mentioned various genes and proteins. Examples thereof include cell, tissue, urine, blood and the like taken from a body. Preferable biological samples are a tissue from ileum or colon, that permits confirmation of marked potentiation of the expression, more preferably a tissue from colon. Then, an mRNA or a protein is extracted from the sample. When an mRNA is extracted, an expression thereof is examined using the diagnostic reagent of the present invention which contains a probe, according to a method generally employed in the pertinent field, such as northern blot and the like. It is also possible to conduct RT-PCR and the like using the diagnostic reagent of the present invention which contains a primer pair. When a protein is extracted, an expression thereof is examined using the diagnostic reagent of the present invention which contains an antibody or a fragment thereof, according to a method generally employed in the pertinent field, such as immunoblot, western blot and the like.
Moreover, the presence or otherwise of the lesion observed in Crohn""s disease can be known or the lesion can be identified by preparing a tissue sample from a tissue obtained from a diagnosis target and subjecting the sample to tissue staining using the diagnostic reagent of the present invention which contains a probe or an antibody.
When the expression of the gene or protein examined as mentioned above is high, the animal is diagnosed as having developed Crohn""s disease or having a high likelihood of developing Crohn""s disease. When an accurate judgment is desired, comparison with the expression at the site (e.g., small intestine) expected to show no potentiation of the expression of the above series of genes or proteins, which are characteristic of Crohn""s disease, is desirable.
According to the present invention, the aforementioned diagnostic reagent of the present invention and other reagents necessary for various methods using the inventive reagent, and the like are preferably packaged in combination to give a kit. For example, when the expression at the gene level is to be analyzed, a reagent for isolating the gene from a biological sample, such as surfactant, protease etc., buffer and the like may be contained in the kit. When the expression at the protein level is to be analyzed, for example, a reagent for extracting the protein from a biological sample, buffer and the like, and where necessary, a secondary antibody, a color developer reagent and the like may be contained in the kit.
It is also possible to construct a screening system of a pharmaceutical agent useful for Crohn""s disease using the diagnostic reagent of the present invention and the principle of the diagnostic method of the present invention.
For this screening system, any cell population can be used as long as it can be treated with a pharmaceutical agent and it affords observation of changes in the expression of the aforementioned gene or protein specific to Crohn""s disease. Specifically, a tissue derived from a target animal, animal other than human (mouse, rabbit and the like), cells of various primary cultures or established cell lines and the like are used. This tissue is appropriately determined depending on the main expression site of the target gene. A tissue is obtained or prepared from a given animal, treated with a given pharmaceutical agent for screening, and expression of at least one or all of PP6 regulated by IL-2, its gene, TNIK, its gene, FLIP, its gene, GRxcex1, its gene, cytochrome oxidase subunit I, its gene, cytochrome b and its gene in the tissue is analyzed. The expression at the protein level and that at the gene level can be determined in parallel, whereby the action mechanism of the pharmaceutical agent for the screening can be postulated.
The present invention is explained in detail by referring to examples. The present invention is not limited by these examples in any way.